7 protocols
AccessionType
normalization data transformation protocol
The 4 in-slide replicates from each slide were combined and the resulting list containing 20 replicates for each gene were normalized such that the average normalized ratio of medians of each spot in the combined list was equal to one. The data sets were then subjected to statistical analysis using Significance Analysis of Microarray (SAM) software under an academic license from Stanford University. Delta values were chosen such that the false discovery rates were between 4 - 5% for each data set and were 0.727, 0.799, 0.748, 0.58 for heat stress, oxygen stress, hypo-tonic stress and blood, respectively. Up- and downregulated genes were extrapolated from significance lists generated by SAM by identifying the average ratio of median value of the replicates for each gene and selecting genes that had values either above 1.8 or below 0.55. Fold regulation shown is the average ratio of median value for each gene.
array scanning protocol
Fluorescent intensities of each spot were calculated using Genepix Pro, version 6.0 (MDS). The program’s morphological opening background subtraction was used to reduce noise and each array was normalized such that the average normalized ratio of medians was equal to one.
(Parameters: Scanning hardware = GenePix 4000A [Axon Instruments], Scanning software = GenePix Pro [Axon Instruments])
nucleic acid hybridization to array protocol
Microarrays were obtained through the NIAID’s Pathogen Functional Genomics Resource Center, managed and funded by the Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS and operated by the J. Craig Venter Institute. Each microarray experiment was performed with control cDNA labeled with Cy3 and test cDNA labeled with Cy5. One array for each condition was used in a dye-swapping experiment to address the possible effects of labeling bias. Freshly purified labeled test and control cDNA were combined prior to incubation with hybridization solution (1x: 3x SSC, 24 mM HEPES (pH 7.0), 0.225% SDS) at 95°C for 2 min. Samples were then evenly dispersed onto microarray slides with cover-slips by capillary action. Hybridization chambers were sealed and incubated at 48°C for 12 hrs. Labeled arrays were washed twice with 3 sequential solutions for 10 min each. Solution 1 (low stringency) contained 2x SSC and 0.1% SDS and was heated to 55°C prior to washing the slides. Solution 2 (medium stringency) contained 0.1x SSC and 0.1% SDS. Solution 3 (high stringency) contained 0.1x SSC. Slides were briefly washed with water prior to drying and scanning on a Genepix 4000A scanner (MDS, Sunnyvale California).
(Parameters: Chamber type = Corning Microarray Technology- CMT-Hyb chamber, Quantity of label target used = 10, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 48)
nucleic acid labeling protocol
For all microarray experiments, 5 ?g of control or experimental RNA was combined with 10 ?g of random hexamers and hybridized at 70°C for 10 min. The following was added with final concentrations listed: 1x Superscipt buffer (Invitrogen), 100 µM DTT, 200 U of SuperScript® III Reverse Transcriptase (Invitrogen), 500 µM each of dATP, dCTP, and dGTP with 300 µM dTTP and 200 µM 5-(3-aminoallyl)-dUTP (Ambion). The reactions were then incubated in a thermocyler at 37°C for 10 min, 42°C for 1 hr 50 min, then 50°C for 10 min. RNA was hydrolyzed in the presence of 0.1 M EDTA and 0.2 N NaOH at 65°C for 10 min. A final concentration of 0.3 M Hepes pH 7.5 was added to buffer the reactions. cDNA was further purified and concentrated using Microcon-30 filters (Millipore) and sodium bicarbonate (pH 9.0) was added to a final concentration of 0.1 M. Amersham mono-reactive Cy™3 and Cy™5 (GE Healthcare) dyes were diluted in DMSO and incubated with the corresponding cDNA samples in the dark for 1 hr at room temperature. Labeled cDNA was then purified with Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer’s protocol.
(Parameters: Amount of nucleic acid labeled = 5, Amplification = none, Mass unit = Micro gram)
nucleic acid hybridization to array protocol
Microarrays were obtained through the NIAID’s Pathogen Functional Genomics Resource Center, managed and funded by the Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS and operated by the J. Craig Venter Institute. Each microarray experiment was performed in quadruplicate with control cDNA labeled with Cy3 and test cDNA labeled with Cy5. One array for each condition was used in a dye-swapping experiment to address the possible effects of labeling bias. Microarray experiments involving blood were done in quadruplicate with 2 dye-swapping slides. Freshly purified labeled test and control cDNA were combined prior to incubation with hybridization solution (1x: 3x SSC, 24 mM HEPES (pH 7.0), 0.225% SDS) at 95°C for 2 min. Samples were then evenly dispersed onto microarray slides with cover-slips by capillary action. Hybridization chambers were sealed and incubated at 48°C for 12 hrs. Labeled arrays were washed twice with 3 sequential solutions for 10 min each. Solution 1 (low stringency) contained 2x SSC and 0.1% SDS and was heated to 55°C prior to washing the slides. Solution 2 (medium stringency) contained 0.1x SSC and 0.1% SDS. Solution 3 (high stringency) contained 0.1x SSC. Slides were briefly washed with water prior to drying and scanning on a Genepix 4000A scanner (MDS, Sunnyvale California).
(Parameters: Chamber type = Corning Microarray Technology- CMT-Hyb chamber, Quantity of label target used = 10, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 48)
growth protocol
Treponema denticola ATCC 35405, Fusobacterium nucleatum ATCC 23726, Streptococcus sanguinis ATCC 10556 and Porphyromonas gingivalis W83 were cultivated in TYGVS medium [15], while Tannerella forsythia ATCC 43037 was grown in new oral spirochete (NOS) medium supplemented with vitamin K (0.2 ?g/ml) and N-acetylmuramic acid (0.01 ?g/ml) [16] . Cells were grown in either 15ml or 50ml centrifuge tubes in an anaerobic chamber (5% CO2, 5% H2 and 90% N2) at 37°C. Co-incubation experiments were performed as follows: 5x109 cells of each bacterium were pelleted at 4,600xg for 10 minutes at room temperature then resuspended in 5ml of pre-reduced TYGVS. Bacteria were then combined such that T. denticola was paired with each individual bacterium . Dual species suspensions were then pelleted again at 4,600xg for 10 minutes at room temperature, replaced into the anaerobic chamber, and incubated at 37°C for 5 hours. One 20ml tube of “unpaired” T. denticola was treated identically and diluted 1:1 in pre-reduced TYGVS as a control. All experiments were performed in triplicate in 15 ml tubes for 5 hours.
(Parameters: start time = 5, time unit = hours, min temperature = 37, temperature unit = C, media = TYGVS or Modified NOS)
nucleic acid extraction protocol
Following the five hour incubation , supernatants were removed from pelleted bacteria. T. denticola RNA was extracted using Trizol® Plus Reagent (Invitrogen Corp., CA, USA) according to the manufacturer’s instructions. Extracted RNA was treated with DNAse I (Ambion) to remove residual genomic DNA. RNA samples were then further purified using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Absence of DNA was confirmed using PCR on extracted RNA. To account for RNA contamination from interacting bacteria, RNA from equivalent monocultures of each bacterium was extracted using the same protocol, normalized according to volume and added to appropriate control samples of T. denticola. Nevertheless, F. nucleatum RNA constituted the overwhelming majority of extracted RNA using this method, as evidenced by the dearth of RNA extracted from monocultures of other bacteria.
(Parameters: Extracted product = total_RNA, Amplification = none)